Callus cultures were established from cotyledon explants from mature fruits of bael cvs. viz., Local, Gonda Selection and Mirzapuri on Knop''s medium supplemented with different combinations of phytohormones.
Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.907.177 Recent Advances in Plant Regeneration from Callus Cultures of Bael [Aegle marmelos (L.) corr.] and Projection of its Economic and Social Benefits Murari Lal, Gulab Singh, DK Sharma, Kanta Sabharwal, Neelam Kumari* and Ravinder* Krishi Vigyan Kendra (KVK), Bhiwani, Ch Charan Singh Haryana Agricultural University (CCS HAU), Hisar-125004, Haryana (India) *Corresponding author ABSTRACT Keywords Plant regeneration, Callus, Cotyledon, Explant, Knop’s medium, Phytohormones, Plantlets, Aegle marmelos Article Info Accepted: 14 June 2020 Available Online: 10 July 2020 Callus cultures were established from cotyledon explants from mature fruits of bael cvs viz., Local, Gonda Selection and Mirzapuri on Knop's medium supplemented with different combinations of phytohormones The maximum callus induction (76.66%) were observed in cv Gonda Selection on Knop's medium supplemented with 2mgl-1 NAA, 2.0mgl-12,4-D & 0.5 mgl-1KIN However, maximum organogenesis (62.50) was observed in cv Local on Knop's medium supplemented with 1.0mgl-1 BAP Maximum number of plantlets per calli (3.85) was observed in cv Mirzapuri from cotyledon derived callus on Knop's medium containing 1.0 mgl-1BAP No plantlets were observed in all the three cvs on Knop's basal medium Introduction The Bael (Aegle marmelos corr.) belongs to the family Rutaceae, to which also belong wood apple, lemon and oranges It is also called Bengal quince The bael fruit has a hard shell and the pulp contains funnels which are filled with mucilage The leaves are astringent, febrifuge, expectorant, and are reported to have hypoglycaemic and antiasthmatic properties (Nambiar, et al., 2000) It is native to India and grows wild all over India The bael is considered to have many medicinal properties and is effective in the treatment of dysentery It also makes an excellent of squash The fruits are a good source of minerals and vitamins (Morton, 1987) and all parts of the plant (i.e stem, bark, roots, leaves and fruit) are used in Ayurvedic medicine (Jayaweera, 1982) The unripe and ripe fruits are useful for curing diarrhoea, dysentery, and stomachalgia (Warrier et al., 1996) It can stand on swampy and alkaline soil and is propagated usually by seeds, root suckers and budding Seedlings have long phase of juvenility and the first 1534 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 crop is obtained very late Root suckers formations is rare occurrence and the budding which is slow, difficult and season bound Micro propagation techniques have been widely used for the propagation of several plant species during the past years Protocols have also been developed for in vito propagation of a number of fruit trees (Bajaj, 1986; Hutchinson and Zimmerman, 1987) Arya et al., (1981) reported callus formation and some organogenesis from cultures developed from cotyledon and hypocotyl explants Plant regeneration via in vitro methods has been reported in A marmelos from different explants, i.e., cotyledonary node (Nayak et al., 2007), root segments (Bhati et al., 1992), nucellus (Hossain et al., 1994), and single-node segments (Ajithkumar and Seeni, 1998) Therefore, attempts have been made for rapid multiplication of bael cvs through callus culture form cotyledon explants thoroughly washed in sterilized single distilled water to remove all the traces of mercuric chloride All the disinfection operations were carried out in the horizontal laminar flow cabinet Culture media The Knop’s (1865) basal medium with some modifications was used for callus induction as well as plant regeneration Cotyledon explants without embryo axis cultured on Knop’s medium supplemented with 3%(w/v) sucrose 100mgl-1 inositol and different combinations of BAP, KIN, NAA and 2,4-D The data on per cent explants showing callus induction, organogenesis and mean number of plantlets through intermediate stage of callus were recorded after seven weeks of culture initiation The media were melted at 1.2kg cm-2 pressure for 15 minutes in the autoclave Then the culture bottles with lid and the flasks were closed with cotton plugs and sterilized Culture conditions Materials and Methods Plant material Cotyledon explants were excised from mature fruits which were collected form 14-15 years old trees of bael cvs Viz., Local, Gonda Selection and Mirzapuri growing at experimental orchard of the Department of Horticulture, CCS Haryana Agricultural University, Hisar The explants were collected and culture in the months of December, January and February (2001-2002) Seeds were separated from the fruits and thoroughly washed in tap water with few drops of teepol Cotyledons were removed from seeds and 47mm diameter cotyledon explants were prepared by removing embryo axis and then treated with 70 per cent aqueous solution of ethanol (v/v) for 30 seconds followed by 0.1 per cent aqueous solution of HgCl2 (w/v) for minutes Finally, the explants were All the cultures were maintained at 25 ±20C under 16/8 hours cycle of light (2000 lux fluorescent tubes) Callus culture Callus was induced by culturing cotyledon explants on Knop’s medium supplemented with various combinations of BAP, KIN, NAA and 2, 4-D Small portions (50-60mg) of the primary callus that green from the cut end and the abaxial surface were removed from the oringinal explants and subcultured into fresh medium after every 20-25 days of callus growth and maintenance of callus cultures Production of in vitro plants Number of plantlets obtained through intermediate stage of callus was transferred to 1535 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 half and full strength Knop2s medium supplemented with different concentrations and combinations of growth regulators Results and Discussion Callus Induction Callus induction was observed in cotyledon explants on various media formulations within 10-21 days of inoculation Callus induction was seen usually at cut end and on the abaxial surface of explants On the basis of all the three cvs used in the present investigation, cv Gonda Selection showed maximum callus induction (76.66%) on K17 medium followed by (74.44%)on K15 medium in cv Mirzapuri whereas, cv Local showed maximum callus growth (66.66%)on K15 and K16 media Minimum callus growth (25.55%) was recorded on K13medium in cv Mirzapuri No callusing was observed on K0 medium in all the three cvs (Fig & Table 2) The growth regulators requirements for callus initiation and organogenesis varied between the three cvs were studied Standardization of media compositions for callus induction and establishment of cultures of the experiment crops is first step in the application of any tissue culture technique The specific combination and concentration of growth regulators Nutrients and incubation conditions modify the normal physiology of explants and induce de-differentiation and redifferentiation of tissues Thus, it is necessary to understand the nutrient requirements and physical factors influencing callus induction In the present investigation, callus induction was observed in all the media tested except Knop’s medium without growth regulators in bael Cotyledon explants after seven weeks of culture revealed maximum percentage of callus induction in cv Gonda section on Knop's medium supplemented with 0.5mgl-1 KIN, 2.0mgl-1 NAA & 2.0 mgl-1 2, 4-D followed by cv Mirzapuri on Knop's medium containing 0.2 mgl-1 BAP & 1.0 mgl1 2, 4-D (Fig and Table 2) Hossain et al., (1994b) reported maximum callus (95.2%) on MS medium supplemented with mgl-1NAA and mgl-1 KIN The differential response is due to difference between media with different combinations of growth regulators Moreover, the choice of genotype beside explant is also very important which plays a definite role in callus induction M.S Hazeena and G.R Sulekha (2008) reported that Murashige and Skoog (MS) medium supplemented with benzyl adenine (2.2 μM) and 2,4-dichlorophenoxy acetic acid (2.26 μM) recorded the highest growth score for callus induction and proliferation Pranita Jamdhade and Narayan Pandhure (2016) observed maximum callus induction on Murashige and Skoog (MS) medium supplemented with 2,4-D (2.0 mg/l) alone and combination of 2,4-D (0.5 mg/l) +KIN (2.0mg/l) + NAA (1.0 mg/l) Per cent Organogenesis from various calli On the basis of all the three cvs used in the present investigation, cv Local showed maximum organogenesis (62.50%) on K3 medium followed by cv Mirzapuri (57.14%) on K12 medium (Refer Plate & 2) whereas, Gonda Selection showed maximum organogenesis (50.00%) on K3 and K12 Media Minimum organogenesis (12.50%) was observed in cv Gonda Selection on K7 medium in Fig and Table The responses of different media combination on number of plantlets obtained through intermediate stage of callus have been presented in Fig and Table On the basis of results, it is clear that among three cvs., cv Mirzapuri (refer Plate 3) showed maximum mean number of plantlets per calli (3.85) on K12 medium followed by 3.42 and 3.00 in cvs Local and Gonda Selection on same media However, minimum mean numbers of plantlets per calli (0.66) were observed in cv Mirzapuri on K10 1536 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 medium The data on mean shoot length using cotyledon explants of different cvs on different combination of media are presented in Fig and Table Among all the three cvs maximum mean shoot length (1.54 cm) was observed in cv Local on K3 medium followed by cv Mirzapuri (1.53cm) and Gonda Selection (1.36cm) on K1 and K3 media respectively Minimum mean shoot length (0.83) was observed in cv Gonda Selection on K7 medium Percentages of rooting in plantlets obtained through intermediate stage of calli are presented in Fig and Table Regeneration of complete plants from single callus and tissues is of great importance for the application of biotechnology in crop improvement Application of biotechnology techniques (in vitro mutant selection and protoplast fusion to cell culture) had limitation in many crop species because of the instability to regenerate plants Only few species have been exploited fully to such studies In present organogenesis studies, among all the three cvs., cv Local showed best organogenesis on Knop's medium containing 1.0 mgl-1 BAP followed by cv Mirzapuri at 1.0Mgl-1 BAP,1.0 mgl-1 KIN & 0.5 mgl-1 NAA No organogenesis was observed on Knop's basal medium (Fig and Table 2) Table.1 List of media tested for callus induction/ shoot regeneration in different explants of bael xop Media Basal medium 10 11 12 13 14 15 16 17 18 K0 K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 K15 K16 K17 Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Knop’s Growth regulators (mgL-1) Cytokinin Auxin BAP KIN NAA 2,4-D 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.50 0.00 0.00 0.00 1.00 0.00 0.00 0.00 2.00 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.50 0.00 0.00 0.00 1.00 0.00 0.00 0.00 2.00 0.50 0.00 0.50 0.50 0.50 0.00 0.50 1.00 0.50 0.00 1.00 0.50 0.50 0.00 0.20 1.00 0.50 0.00 0.20 0.00 0.00 0.20 0.20 0.00 0.00 0.50 0.00 0.00 0.00 1.00 0.50 0.50 5.00 0.00 0.50 0.50 2.00 2.00 1537 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 Table.2 Effect of different media combination on in vitro callus induction of bael cvs from cotyledon explants Media K0 K13 K14 K15 K16 K17 Local 54.44±2.93 65.55±2.93 62.22±2.22 48.88±1.11 57.77±2.22 Per cent shoot regeneration Gonda selection Mirzapuri 0 51.11±1.11 25.55±2.93 51.11±5.87 51.11±1.11 56.66±3.33 74.44±2.93 63.33±3.33 45.55±2.93 76.66±3.33 56.66±3.33 ± SE Table.3 Effect of different media combination on per cent Organogenesis from various calli induced of bael cvs Media K0 K2 K3 K6 K7 K10 K12 Local 42.85 62.50 28.57 37.50 42.85 57.14 Per cent organogenesis Gonda Selection Mirzapuri 0 28.57 33.33 50.00 50.00 14.28 16.66 12.50 33.33 28.57 16.66 50.00 57.14 ± SE Table.4 Effect of different media combination on mean number of plantlets from various calli induced of bael cvs Media K0 K2 K3 K6 K7 K10 K12 Mean number of plantlets per calli Local Gonda selection Mirzapuri 0 2.28±1.12 1.28±0.89 2.00±1.29 2.37±0.80 3.25±1.46 2.00±1.36 1.57±1.06 1.14±1.14 0.83±0.83 2.12±1.14 1.12±1.12 1.66±1.17 1.71±0.91 1.85±1.24 0.66±0.66 3.42±1.39 3.00±1.19 3.85±1.66 ± SE 1538 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 Table.5 Effect of different media combination on mean shoot length from various calli induced of bael cvs Media K0 K2 K3 K6 K7 K10 K12 Local 1.20±0.22 1.54±0.15 1.21±0.18 1.17±0.17 1.32±0.20 1.32±0.14 Mean shoot length(cm) Gonda selection 0.97±0.18 1.36±0.16 0.87±0.13 0.83±0.10 1.14±0.16 1.18±0.10 ± SE 1539 Mirzapuri 1.26±0.18 1.47±0.19 1.33±0.35 1.53±0.21 1.01±0.37 1.51±0.14 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 Arya et al., (1981) reported shoot development from meristemoids occurred only when they were transferred to BAP and KIN alone or BA+NAA, whereas KIN was quite suitable for shoot induction from cotyledonary explant callus in bael Generally, low auxin and high cytokinin concentration in the medium resulted in induction of shoot morphogenesis in the present study M.S Hazeena and G.R Sulekha (2008) reported that shoot regeneration response from the callus was best on MS medium containing 8.8 μM benzyl adenine and 2.85 μM indole-3- acetic acid Pranita Jamdhade and Narayan Pandhure (2016) was observed maximum induction of somatic embryogenesis and shoot induction from cotyledon explant on Murashige and Skoog (MS) medium supplemented with BAP(2.0 mg/l) + NAA (0.5mg/l) 1540 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 1534-1542 Number of plantlets obtained through intermediate stage of callus Maximum mean number of plantlets per calli were observed in cv Mirzapuri from cotyledon explant derived callus on Knop's medium having 1.0 mgl-1 BAP, 1.0 mgl-1 KIN & 0.5 Mgl-1 NAA followed by cv Local on media containing 1.0mgl-1BAP, 1.0 mgl-1 KIN & 0.5 mgl-1 NAA and 1.0 mgl-1 BAP No plants were observed in all the three cvs on Knop's basal medium (Fig.3 and Table 4) It might be due to higher concentration of cytokinin which enhances the shoot initiation and auxin elongates the shoot primordial Varghese et al., (1993) reported that low concentration of BAP resulted in less development of shoots (3.2), but higher concentration promoted greater number (11.82) of shoots However, BAP in combination with NAA resulted in the percentage of shoot formation in bael Similarly, Rao and Lee (1982) achieved multiple shoot formation in calophyllum, Eugenia and Fragaria in presence of BAP This was in contrast to the present investigation could be due to differences in genotypes and variation in endogenous level of growth regulators in cotyledon explants Mean shoot length Knop's medium supplemented with 1.0 mgl-1 BAP produced better shoot length in cvs Local and Mirzapuri (Fig and Table 5) Hossain et al., (1994a) reported maximum mean shoot length (7.2) on MS medium supplemented with 5mgl-1 BAP+0.1mgl-1 NAA+1.0mgl-1 GA3 in bael This difference could be genotype and growth regulators used Acknowledgement Thanks to Chaudhary Department of Horticulture Charan Singh Haryana Agricultural University (CCSHAU),Hisar; Dr Suneel Sharma former Professor, CCSHAU, Hisar; Dr S.S.Sindhu former Professor, CCSHAU, Hisar; Dr R S Huda, Director, Directorate of Extension Education, CCSHAU, Hisar; Dr Anil Godara Prof & Head, Department of Horticulture, CCSHAU, Hisar for providing facilities for this study References Arya, H C., Ramawat, K G., & Suthar, K C 1981 Culture and differentiation of plants of economic importance II Aegle marmelos L J Indian Bot Soc, 60: 134137 Ajithkumar, B., D and Seeni, S.1998 Rapid clonal multiplication through in 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Corr Via organogenic callus culture Cey J Sci (Bio Sci.), 35 (1): 87-90 Rao, A N., & Lee, S K 1982 Importance of tissue culture in tree propagation In Plant tissue culture 1982: proceedings, 5th International Congress of Plant Tissue and Cell Culture held at Tokyo and Lake Yamanake, Japan, July 11-16, 1982/edited by Akio Fujiwara Tokyo: Japanese Association for Plant Tissue Culture Varghese, S K., Inamdar, J A., Kalia, K., Subramanian, R B., & Nataraj, M 1993 Micropropagation of Aegle marmelos (L) Corr Phytomorphology, 43 (1-2): 87-92 Warrier, P.K., Nambiar, V.P.K., and Ramankutty, C 1996 Indian Medicinal Plants Vol Orient Longman, Madras, pp 62–66 How to cite this article: Murari Lal, Gulab Singh, DK Sharma, Kanta Sabharwal, Neelam Kumari and Ravinder 2020 Recent Advances in Plant Regeneration from Callus Cultures of Bael [Aegle marmelos (L.) corr.] and Projection of its Economic and Social Benefits Int.J.Curr.Microbiol.App.Sci 9(07): 1534-1542 doi: https://doi.org/10.20546/ijcmas.2020.907.177 1542 ... Kumari and Ravinder 2020 Recent Advances in Plant Regeneration from Callus Cultures of Bael [Aegle marmelos (L.) corr.] and Projection of its Economic and Social Benefits Int.J.Curr.Microbiol.App.Sci... subcultured into fresh medium after every 20-25 days of callus growth and maintenance of callus cultures Production of in vitro plants Number of plantlets obtained through intermediate stage of callus. .. whereas KIN was quite suitable for shoot induction from cotyledonary explant callus in bael Generally, low auxin and high cytokinin concentration in the medium resulted in induction of shoot